Methods for detecting and treating cancer

ABSTRACT

Methods and kits for detecting cancer and monitoring cancer progression are described. The method involves analyzing a sample containing nucleic acids or proteins from a patient for decreased expression of endoglycan and/or increased expression of podocalyxin.

FIELD OF THE INVENTION

The invention relates to methods and kits for detecting and monitoringthe progression of cancer, in particular breast cancer. The inventionalso includes methods of treating cancer.

BACKGROUND OF THE INVENTION

Metastatic breast cancer is the leading cause of death among womenbetween the ages of 15 and 54 and affects approximately 13% of womenduring their lifespan. These can be grossly categorized as ductal orlobular depending on their site of origin in normal breast tissue.Tumors usually begin as non-invasive cells at the site of tumor origin,spread to surrounding tissue in the breast and eventually become fullymetastatic and migrate to the lymph nodes and other parts of the body.

There is increasing evidence that cell-cell adhesion is a potentsuppressor of metastatic breast cancer progression (Berx and Van Roy,2001). For example, in infiltrating lobular breast carcinomas E-cadherinis often lost and the resulting disruption of adherens junctionsinitiates a complete dissolution of cell-cell adhesion which allowssingle cells to break away from the primary tumor and invade the stromain a single file pattern (Cleton Jansen et al., 2002). Alterations incell adhesion are more subtle in infiltrating ductal carcinomas whereinvasion is characterized by the movement of clusters of cells into thestroma (Page and Simpson, 2000). In the latter situation adherensjunctions are often present (Acs et al., 2001; Gillett et al., 2001) butthere appears to be a general loss of polarity that is characterized bythe mislocalization of apical markers such as MUC-1-(McGuckin et al.,1995; Mommers et al., 1999; Diaz et al., 2001; Rahn et al., 2001) thatmay be fueled by the disruption of tight junctions (Hoover et al., 1997;Kramer et al., 2000; Kominsky et al., 2003). While transcriptionalrepressors of E-cadherin expression have been identified (Cano et al.,2000; Guaita et al., 2002), little is known about the mechanismresponsible for the disruption of tight junctions during breast tumorprogression.

CD34 was initially identified over 20 years ago as an hematopoietic stemcell and vascular endothelial marker and has alternatively been proposedto act as an: 1) enhancer of proliferation, 2) a blocker ofdifferentiation, 3) bone marrow homing receptor, 4) cell adhesionmolecule, and 5) a blocker of cell adhesion (Fackler et al, 1996, Krauseet al. Blood, 1996, Baumhueter et al. 1993). Deletion of the CD34 genein mice has only served to fuel this debate as these mice are relativelynormal with very subtle defects in hematopoietic and vascular function.The most clear-cut experiments suggest that CD34-type proteins can actas either pro-adhesive or anti-adhesive molecules depending on theirglycosylation status (Satomaa, 2002, Baumhueter et al., 1993 and Bistrupet al., 1999).

Podocalyxin, (also called podocalyxin-like protein 1 (PCLP-1),Myb-Ets-transformed progenitor (MEP21) or thrombomucin) is a heavilysialylated and sulfated integral membrane glycoprotein that interactswith the actin cytoskeleton. It belongs to the CD34 family ofsialomucins and is highly expressed on the surface of hematopoeiticprogenitors, vascular endothelia and podocytes which form a tightjunction-free epithelial meshwork that surrounds glomerular capillariesin the kidney (Kerjaschki et al., 1984; Kershaw et al., 1995; McNagny etal., 1997). Evidence suggests that the primary function of this moleculeis to act as a type of molecular “Teflon ™” to block inappropriate celladhesion. For example, as kidney podocytes begin to express podocalyxinthey undergo a dramatic morphological shift from adherent, tightjunction-associated monolayers surrounding the glomerular capillaries toa more modified cell layer lacking tight junctions and with extensivefully-interdigitated foot processes that are separated from each otherby slit diaphragms. These podocalyxin-covered slit diaphragms form theprimary filtration apparatus of the kidney. Deletion of thepodocalyxin-encoding gene in mice results in the persistence oftight-junctions between podocytes, a lack of foot process formation andperinatal death due to anuria and high blood pressure (Doyonnas et al.,2001). Conversely, when podocalyxin is ectopically expressed in kidneyepithelial cell monolayers, tight junctions and adherens junctions areboth disrupted (Takeda et al., 2000). Thus, both gain-of-function andloss-of-function experiments suggest that podocalyxin acts as atissue-specific anti-adhesin during normal kidney development (Takeda etal., 2001, Doyonnas et al., 2001).

Circumstantial evidence suggests that podocalyxin expression may beupregulated in a variety of neoplastic scenarios. For examplepodocalyxin was recently identified as the peanut agglutinin-bindingtumor antigen gp200 expressed on human embryonal carcinomas. (Schopperleet al., 2002). In addition, the human podocalyxin gene (PODXL) has beenassigned to chromosome 7q32-q33 (Kershaw et al., 1997), which placesPODXL very close to the 7q35ter region that has been identified as again site by comparative genomic hybridization in ductal carcinoma insitu, infiltrating ductal carcinoma and in lymph node metastasis (Aubeleet al., 2000). Thus, while it is not yet clear whether the PODXL gene isamplified in breast carcinoma, its expression may be unduly influencedby a nearby amplicon. Under anemic conditions the inventors haverecently shown that Podocalyxin expression is upregulated in mouseerythroid progenitor cells (McNagny submitted unpublished obs).Therefore, podocalyxin expression may be similarly upregulated innecrotic breast carcinomas where hypoxia-regulated genes aretranscriptionally activated (Adeyinka et al., 2002). If this is indeedthe case, it would have functionally important implications as tumorhypoxia helps to drive solid tumor progression generally (Knowles andHarris, 2001) and ductal carcinoma progression specifically (Bos et al.,2003; Helczynska et al., 2003).

Using homologies present in the cytoplasmic tails of CD34 andpodocalyxin, endoglycan was identified as a novel member of this familyof glycoproteins. Endoglycan mRNA and protein were detected in bothendothelial cells and CD34+ bone marrow cells (Sassetti et al., 2000).Endoglycan, like CD34 and podocalyxin can function as a L-selectinligand. Endoglycan utilizes a different binding mechanism, interactingwith L-selectin through sulfation on two tyrosine residues and O-linkedsLex structures (Fieger et al., 2003).

SUMMARY OF THE INVENTION

The inventors have shown that podocalyxin is a prognostic indicator oftumor metastasis and that it plays an active role in making cells lessadherent and more invasive. The present inventors have also shown thatendoglycan is an antagonist of podocalyxin.

Accordingly, in one embodiment, the present invention provides a methodfor detecting cancer in a patient comprising:

(a) determining the level of podocalyxin in a sample from the patient;and

(b) comparing the level of podocalyxin in the sample to a controlsample, wherein increased levels of podocalyxin as compared to thecontrol indicates that the patient has cancer.

In another embodiment, the present invention provides a method fordetecting cancer in a patient comprising:

(a) determining the level of endoglycan in a sample from the patient;and

(b) comparing the level of endoglycan in the sample to a control sample,wherein decreased levels of endoglycan as compared to the controlindicates that the patient has cancer.

In a further embodiment, the present invention provides a method fordetecting cancer in a patient comprising:

(a) determining the level of endoglycan and podocalyxin in a sample fromthe patient; and

(b) comparing the ratio of endoglycan to podocalyxin in the sample to acontrol sample, wherein a decreased ratio of endoglycan to podocalyxinas compared to the control indicates that the patient has cancer.

In yet another embodiment, the present invention provides a method formonitoring the progression of cancer in a patient, comprising:

(a) determining the level of podocalyxin in a sample from the patient;and

(b) repeating step (a) at a later point in time and comparing the resultof step (a) with the result of step (b) wherein a difference in thelevel of podocalyxin expression is indicative of the progression of thecancer in the patient.

In another embodiment, the present invention provides a method formonitoring the progression of cancer in a patient, comprising:

(a) determining the level of endoglycan in a sample from the patient;and

(b) repeating step (a) at a later point in time and comparing the resultof step (a) with the result of step (b) wherein a difference in thelevel of endoglycan expression is indicative of the progression of thecancer in the patient.

In a further embodiment, the present invention provides a method formonitoring the progression of cancer in a patient comprising:

(a) determining the level of endoglycan and podocalyxin in a sample fromthe patient; and

(b) repeating step (a) at a later point in time and comparing the resultof step (a) with the result of step (b) wherein a difference in theratio of endoglycan to podocalyxin is indicative of the progression ofthe cancer in the patient.

In another embodiment, the present invention provides a method fordetermining whether or not a cancer is metastatic in a patientcomprising:

(a) detecting the level of podocalyxin in a sample from the patient; and

(b) comparing the level of podocalyxin in the sample to a controlsample, wherein increased levels of podocalyxin as compared to thecontrol indicates that the cancer is metastatic.

In yet another embodiment, the present invention provides a method fordetermining whether or not a cancer is metastatic in a patientcomprising:

(a) detecting the level of endoglycan in a sample from the patient; and

(b) comparing the level of endoglycan in the sample to a control sample,wherein decreased levels of endoglycan as compared to the controlindicates that the cancer is metastatic.

In a further embodiment, the present invention provides a method fordetermining whether or not a cancer is metastatic in a patientcomprising:

(a) detecting the level of endoglycan and podocalyxin in a sample fromthe patient; and

(b) comparing the ratio of endoglycan to podocalyxin in the sample to acontrol sample, wherein a decreased ratio of endoglycan to podocalyxinas compared to the control indicates that the cancer is metastatic.

In preferred embodiments of the invention, the above methods are used todetect breast cancer.

The present invention includes methods of treating cancer by modulating,preferably inhibiting, the levels of podocalyxin on the cancer. Theapplication also includes methods for the identification of compoundsthat modulate the biological activity of podocalyxin that may be usedfor the treatment of cancers with increased expression of podocalyxin.

The present invention includes methods of treating cancer by modulating,preferably agonizing, the levels of endoglycan on the cancer. Theapplication also includes methods for the identification of compoundsthat modulate the biological activity of endoglycan that may be used forthe treatment of cancers with decreased expression of endoglycan.

Accordingly, the present invention provides a method of modulatingcancer cell growth by administering an effective amount of an agent thatmodulates endoglycan and/or podocalyxin to a cell or animal in needthereof.

The present invention also includes screening assays for identifyingagents that modulate endoglycan and/or podocalyxin and that are usefulin modulating cancer cell growth. Agents that modulate include agentsthat stimulate (agonists) and agents that inhibit (antagonists).

Accordingly, the present invention provides a method for identifying acompound that modulates podocalyxin comprising:

(a) incubating a test compound with podocalyxin or a nucleic acidencoding podocalyxin; and

(b) determining the effect of the compound on podocalyxin activity orexpression and comparing with a control (i.e. in the absence of the testsubstance), wherein a change in the podocalyxin activity or expressionas compared to the control indicates that the test compound modulatespodocalyxin.

In another embodiment, the present invention provides a method foridentifying a compound that modulates endoglycan comprising:

(a) incubating a test compound with endoglycan or a nucleic acidencoding endoglycan; and

(b) determining the effect of the compound on endoglycan activity orexpression and comparing with a control (i.e. in the absence of the testsubstance), wherein a change in the endoglycan activity or expression ascompared to the control indicates that the test compound modulatesendoglycan.

The present invention includes pharmaceutical compositions containingone or more modulators of endoglycan and/or podocalyxin. Accordingly,the present invention provides a pharmaceutical composition for use inmodulating cancer cell growth comprising an effective amount ofendoglycan/podocalyxin modulator in admixture with a suitable diluent orcarrier.

In one embodiment, the present invention provides a pharmaceuticalcomposition for use in treating cancer comprising an effective amount ofa podocalyxin antagonist in admixture with a suitable diluent orcarrier. In another embodiment, the present invention provides apharmaceutical composition for use in treating cancer comprising aneffective amount of an endoglycan agonist in admixture with a suitablediluent or carrier.

Other features and advantages of the present invention will becomeapparent from the following detailed description. It should beunderstood, however, that the detailed description and the specificexamples while indicating preferred embodiments of the invention aregiven by way of illustration only, since various changes andmodifications within the spirit and scope of the invention will becomeapparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will now be described in relation to the drawings inwhich:

FIG. 1 shows podocalyxin immunostaining of normal tissues and the tumormicroarray. In positive control kidney tissue (A) the podocytes withinthe glomerulus were highly positive while the tubular epithelium wasnegative (see inset). The vascular endothelium of the glomerulus andwithin the kidney cortex was also positive. In normal breast tissue (B)positive staining was observed in the vascular endothelium (arrows) andthe apical regions of luminal breast epithelial cells (see inset;arrowheads). On the tissue microarray invasive breast carcinomas werescored as: ‘0’ (ie. C) if there was no discernable staining on thecarcinoma cells (see inset; positive staining is on endothelial cells);‘1’ (ie. D) if less than 10% of the cells stainined positively; ‘2’ (ie.E) if there was diffuse staining in more than 10% of the cells and/orstrong cytoplasmic staining in less than 50% of the cells; or ‘3’ ifthere was strong cytoplasmic staining in more than 50% of the cells (ie.F).

FIG. 2 consists of two graphs illustrating the prognostic significanceof podocalyxin expression in breast tumors (Kaplan-Meier survivalanalysis). Disease specific survival at all expression levels indicatesthat only the high podocalyxin expression level (+3) is prognosticallysignificant (A). Therefore, expression levels 0 to 2 were combined as“no or low podocalyxin” and +3 as was designated as “high podocalyxin”(B).

FIG. 3 illustrates the functional significance of podocalyxinoverexpression in MCF-7 breast carcinoma cells.

FIG. 3A shows the endogenous levels in three human breast carcinomalines as assessed by Western blotting with an antibody specific forhuman podocalyxin. Note that podocalyxin was modestly expressed inwell-behaved T47D and MCF-7 cells compared to the highly invasive MDA231cell line.

FIG. 3B is a series of photographs showing MCF-7 cells that were controltransfected or stably transfected with an expression vector containingboth GFP and mouse podocalyxin. Control transfected MCF-7 cells formedclassical cobblestone epithelial monolayers (top panel) while bulgingcells were shed from the surface of the GFP/Podocalyxin transfectedcells (middle panel). GFP (green) and mouse podocalyxin (red) werecoordinately expressed in a heterogenous manner (lower panel). (uppertwo panels live phase microscopy, bar=50 μm; lower panel, Z-seriesconfocal dual fluorescence microscopy for GFP and mouse-specificpodocalyxin immunostaining; bar=15 μm).

FIG. 3C is a series of photographs of transfected MCF-7 cells that weretriple stained for mouse podocalyxin (red), DNA/Nuclei (blue) and eitherthe adherens junction protein E-cadherin or the tight junction proteinoccludin (green). Note that where podocalyxin was not expressedE-cadherin was localized basolaterally and occludin was localized atapical terminal bars. In contrast, where podocalyxin was expressed thecells bulged apically (note upward movement of blue nuclei) and bothE-cadherin and occludin localization became depolarized (Z-seriesconfocal microscopy, bar=15 μm)

FIG. 4 shows the CD34 family including their genomic loci, motifs andsplicing. (A) Schematic showing the hypothetical structure of CD34,Podocalyxin, and Endoglycan based on predicted protein sequences andpublished data. Blue boxes=mucin domains, green boxes=the cysteine-richdomains, black circles=potential N-linked carbohydrates, horizontal barswith or without arrows=potential O-linked carbohydrates,arrows=potential sialic acid motifs on O-linked carbohydrates, PKC, CK2and TK=potential phosphorylation sites. (B) Genomic organization ofhuman cd34, podxl and endgl genes based on sequence contigs identifiedin the human sequence database. (C) Alternative splicing of CD34-familytranscripts and their consequences for protein structure. Analyses ofESTs, primary cDNA clones and genomic loci suggest that, for all threefamily members, splicing between exons 7 and 8 results in longer cDNAswith premature translational stops that lead to truncation of thecytoplasmic domains.

FIG. 5 shows homologies between CD34 family orthologs and homologs (SEQID NOS:1-9).

FIG. 6 shows the specificity of rat monoclonal antibody F4B10 toendoglycan compared to other CD34 family members.

FIG. 7 shows reciprocal expression of Endoglycan and Podocalyxin bymetastatic and non-metastatic breast carcinoma lines. FACS profilesshowing Endoglyean and Podocalyxin expression by the metastatic,non-polarized cell, MDA-231 and the non-metastatic, polarized cell lineMCF-7. Green lines=specific antibody staining. Below is a western blotto show relative levels of Podocalyxin in these lines. MCF-7 and asecond non-metastatic line (T47D) express high levels of Endoglycan butlittle if any Podocalyxin. MDA-231, a metastatic line expresses highlevels of Podocalyxin and no Endoglycan.

FIG. 8 shows failure of ectopic Endoglycan expression to block mast cellaggregation. (A) Mast cells from Wild type and cd34^(-/-)/cd43^(-/-)infected with pMXpie retrovirus alone were plated at similar densitiesfor assessment of aggregation. Graphs show data from two independentlyderived bone marrow mast cell cultures. (B) cd34^(-/-)/cd43^(-/-) mastcells infected with pMXpie containing CD34FL, CD34CT or Endoglycan.Graphs show data from two independent infections.

DETAILED DESCRIPTION OF THE INVENTION

I. Diagnostic Methods

The present inventors have determined that podocalyxin is a functionallyimportant molecule in tumor progression. Using a tissue microrray (TMA),the inventors assessed podocalyxin expression and localization in aseries of 270 invasive human breast carcinomas for which fullclinicopathologic follow up and outcome was present. Podocalyxin wasfound to be highly expressed and diffusely distributed in a small subsetof these tumors. It was also found that high podocalyxin expression wasa clear and independent prognostic indicator of poor outcome in thistumor subset. To test the functional consequences of this increasedexpression, murine podocalyxin was ectopically expressed in human MCF-7breast carcinoma cells that normally grow as adherent monolayers withabundant adherens junctions and tight junctions. Low level ectopicpodocalyxin expression lead to the disruption of both adherens and tightjunctions while high cells expressing high levels of the protein werede-polarized and actively extruded from otherwise cohesive MCF-7monolayers. The data demonstrates that podocalyxin is a prognosticindicator of tumor metastasis and that it plays an active role in makingcells less adherent and more invasive. The inventors have also shownthat podocalyxin is involved in decreasing the apical/basal cellpolarity of breast tissues, a hallmark of solid tumor progression. Theinventors have also shown that podocalyxin expression is dramaticallyincreased during hypoxia, as the rapid proliferation of cells duringtumor progression causes the tissue to become hypoxic. Therefore,podocalyxin is a marker of solid tumor progression and a marker of tumorhypoxia.

The present inventors have also determined that endoglycan andpodocalyxin have a mirror image pattern of expression in breast cancercells lines. Endoglycan levels are high in the relatively non-metastaticlines MCF-7 and T47D where podocalyxin levels are low. In contrast,endoglycan expression is negative in the MDA-231 metastatic tumor linecompared to high levels of podocalyxin. Since endoglycan and podocalyxinhave similar sequences in the cytoplasmic domain, endoglycan may be anatural antagonist of podocalyxin. Endoglycan may promote adhesion,maintain cell polarity and block metastasis whereas podocalyxin mayblock adhesion, decrease polarity and increase metastasis. Despiteendoglycan's similarity to CD34 and podocalyxin (FIGS. 4 and 5), it doesnot block cell aggregation when ectopically expressed in CD34/CD43deficient mast cells, a phenotype of ectopic expression of CD34.Podocalyxin is known to bind to the actin cytoskeleton through bindingto NHERF (Li and Kershaw 2002, and Takeda 2001). Since endoglycan bindsNHERF but lacks an anti-adhesive function, it may act as an antagonistof podocalyxin by competing with podocalyxin's ability to interact withthe actin cytoskeleton and more specifically with NHERF.

Accordingly, evaluating endoglycan and/or podocalyxin levels may be usedin the prognostic and diagnostic evaluation of cancers involvingendoglycan and/or podocalyxin, the identification of subjects with apredisposition to such cancers, and in the monitoring of the progress ofpatients with endoglycan related cancers.

In an embodiment of the invention, a method is provided for detectingcancer in a patient comprising:

(a) detecting the level of podocalyxin in a sample from the patient; and

(b) comparing the level of podocalyxin in the sample to a controlsample, wherein increased levels of podocalyxin as compared to thecontrol indicates that the patient has cancer.

In another embodiment of the invention, a method is provided fordetecting cancer in a patient comprising:

(a) detecting the level of endoglycan in a sample from the patient; and

(b) comparing the level of endoglycan in the sample to a control sample,wherein decreased levels of endoglycan as compared to the controlindicates that the patient has cancer.

Evaluating endoglycan levels relative to podocalyxin levels may also beused in the prognostic and diagnostic evaluation of cancers involvingendoglycan, the identification of subjects with a predisposition to suchcancers, and in the monitoring of the progress of patients withendoglycan related cancers.

Accordingly, in another embodiment of the invention, a method isprovided for detecting cancer in a patient comprising:

(a) determining the level of endoglycan and podocalyxin in a sample fromthe patient; and

(b) comparing the ratio of endoglycan to podocalyxin in the sample to acontrol sample, wherein a decreased ratio as compared to controlindicates that the patient has cancer.

The term “podocalyxin” as used herein is synonymous withpodocalyxin-like protein 1 (PCLP-1), Myb-Ets-transformed progenitor(MEP21) or thrombomucin and is a heavily sialyated and sulfated integralmembrane glycoprotein that interacts with the actin cytoskeleton. Theterm podocalyxin includes all of the known podocalyxin moleculesincluding those deposited in GenBank under accession number U97519 orthose referred to in Kershaw et al. (Kershaw D B, Beck S G, Wharram B L,Wiggins J E, Goyal M, Thomas P E, Wiggins R C., Molecular cloning andcharacterization of human podocalyxin-like protein. Orthologousrelationship to rabbit PCLP1 and rat podocalyxin. J Biol Chem. 1997 Jun20;272(25):15708-14) as well as any isoforms, variants, analogs,derivatives or fragments thereof that are useful in detecting cancer.

The term “endoglycan” includes all of the known endoglycan moleculesincluding those deposited in GenBank under accession number AF219137 orthose referred to in Sassetti et al. (Sassefti C, Van Zante A, and S DRosen, (2000) Identification of Endoglycan, a Member of theCD34/Podocalyxin Family of Sialomucins, Journal of Biological Chemistry,275(12):9001) as well as any isoforms, variants, analogs, derivatives orfragments thereof that are useful in detecting cancer.

The phrase “detecting the level of endoglycan” and “detecting the levelof podocalyxin” includes the detection of the levels of protein as wellas detection of the levels of nucleic acid molecules encoding theprotein. Methods for detecting proteins and nucleic acids are discussedin greater detail below.

Endoglycan and podocalyxin are alternatively spliced to give twoisoforms of the protein core; one with a long cytoplasmic tail and onewith a short cytoplasmic tail. Consequently, in a specific embodiment,the methods of the invention are used to detect the short form ofendoglycan and/or podocalyxin.

The term “cancer” as used herein includes all cancers that areassociated with decreased expression of endoglycan and/or increasedexpression of podocalyxin. In a preferred embodiment, the cancer isbreast cancer, more preferably invasive breast carcinoma.

The term “sample from a patient” as used herein means any samplecontaining cancer cells that one wishes to detect including, but notlimited to, biological fluids, tissue extracts, freshly harvested cells,and lysates of cells which have been incubated in cell cultures. In apreferred embodiment, the sample is breast tissue.

The term “control sample” includes any sample that can be used toestablish a base or normal level, and may include tissue samples takenfrom healthy persons or samples mimicking physiological fluid.

The method of the invention may be used in the diagnosis and staging ofcancer, in particular breast cancer. The invention may also be used tomonitor the progression of a cancer and to monitor whether a particulartreatment is effective or not. In particular, the method can be used toconfirm the absence or removal of all tumor tissue following surgery,cancer chemotherapy, and/or radiation therapy. The methods can furtherbe used to monitor cancer chemotherapy and tumor reappearance.

In an embodiment, the invention contemplates a method for monitoring theprogression of cancer in a patient, comprising:

(a) determining the level of podocalyxin expression in a sample from thepatient; and

(b) repeating step (a) at a later point in time and comparing the resultof step (a) with the result of step (b) wherein a difference in thelevel of podocalyxin expression is indicative of the progression of thecancer in the patient.

In particular, increased levels of podocalyxin at the later time pointmay indicate that the cancer is progressing and that the treatment (ifapplicable) is not being effective. In contrast, decreased levels ofpodocalyxin at the later time point may indicate that the cancer isregressing and that the treatment (if applicable) is effective.

In another embodiment, the invention contemplates a method formonitoring the progression of cancer in a patient, comprising:

(a) determining the level of endoglycan expression in a sample from thepatient; and

(b) repeating step (a) at a later point in time and comparing the resultof step (a) with the result of step (b) wherein a difference in thelevel of endoglycan expression is indicative of the progression of thecancer in the patient.

In particular, decreased levels of endoglycan at the later time pointmay indicate that the cancer is progressing and that the treatment (ifapplicable) is not being effective. In contrast, increased levels ofendoglycan at the later time point may indicate that the cancer isregressing and that the treatment (if applicable) is effective.

In a further embodiment, the invention contemplates a method formonitoring the progression of cancer in a patient, comprising:

(a) determining the level of endoglycan and podocalyxin in a sample fromthe patient; and

(b) repeating step (a) at a later point in time and comparing the resultof step (a) with the result of step (b) wherein a difference in theratio of endoglycan to podocalyxin is indicative of the progression ofthe cancer in the patient.

The inventors have also shown that endoglycan and/or podocalyxin is amarker of tumor metastasis. Accordingly, the present invention providesa method of determining whether or, not a cancer is metastatic in apatient comprising:

(a) detecting the level of podocalyxin in a sample from the patient; and

(b) comparing the level of podocalyxin in the sample to a controlsample, wherein increased levels of podocalyxin as compared to thecontrol indicates that the cancer is metastatic.

In another embodiment, the present invention provides a method ofdetermining whether or not a cancer is metastatic in a patientcomprising:

(a) detecting the level of endoglycan in a sample from the patient; and

(b) comparing the level of endoglycan in the sample to a control sample,wherein decreased levels of endoglycan as compared to the controlindicates that the cancer is metastatic.

In a further embodiment, the present invention provides a method ofdetermining whether or not a cancer is metastatic in a patientcomprising:

(a) detecting the level of endoglycan and podocalyxin in a sample fromthe patient; and

(b) comparing the ratio of endoglycan to podocalyxin in the sample to acontrol sample, wherein a decreased ratio of endoglycan to podocalyxinas compared to the control indicates that the cancer is metastatic.

A variety of methods can be employed for the above described diagnosticand prognostic evaluation of cancers involving endoglycan and/orpodocalyxin, and the identification of subjects with a predisposition tosuch disorders. Such methods may rely on, for example, the detection ofnucleic acid molecules encoding endoglycan and/or podocalyxin, andfragments thereof, or the detection of the endoglycan protein and/orpodocalyxin protein using, for example, antibodies directed againstendoglycan and/or podocalyxin, including peptide fragments. Each ofthese is described below.

(a) Methods for Detecting Nucleic Acid Molecules

In one embodiment, the methods of the invention involve the detection ofnucleic acid molecules encoding endoglycan and/or podocalyxin. Thoseskilled in the art can construct nucleotide probes for use in thedetection of nucleic acid sequences encoding endoglycan and/orpodocalyxin in samples. Suitable probes include nucleic acid moleculesbased on nucleic acid sequences encoding at least 5 sequential aminoacids from regions of endoglycan and/or podocalyxin, preferably theycomprise 15 to 30 nucleotides. A nucleotide probe may be labeled with adetectable substance such as a radioactive label which provides for anadequate signal and has sufficient half-life such as ³²P, ³H, ¹⁴C or thelike. Other detectable substances which may be used include antigensthat are recognized by a specific labeled antibody, fluorescentcompounds, enzymes, antibodies specific for a labeled antigen, andluminescent compounds. An appropriate label may be selected havingregard to the rate of hybridization and binding of the probe to thenucleotide to be detected and the amount of nucleotide available forhybridization. Labeled probes may be hybridized to nucleic acids onsolid supports such as nitrocellulose filters or nylon membranes asgenerally described in Sambrook et al, 1989, Molecular Cloning, ALaboratory Manual (2nd ed.). The nucleic acid probes may be used todetect genes, preferably in human cells, that encode endoglycan and/orpodocalyxin. The nucleotide probes may also be useful in the diagnosisof disorders involving an endoglycan and/or a podocalyxin in monitoringthe progression of such disorders; or monitoring a therapeutictreatment. In an embodiment, the probes are used in the diagnosis of,and in monitoring the progression of cancer, preferably breast cancer.

The probe may be used in hybridization techniques to detect genes thatencode endoglycan and/or podocalyxin proteins. The technique generallyinvolves contacting and incubating nucleic acids (e.g. recombinant DNAmolecules, cloned genes) obtained from a sample from a patient or othercellular source with a probe under conditions favorable for the specificannealing of the probes to complementary sequences in the nucleic acids.After incubation, the non-annealed nucleic acids are removed, and thepresence of nucleic acids that have hybridized to the probe if any aredetected.

The detection of nucleic acid molecules may involve the amplification ofspecific gene sequences using an amplification method such as polymerasechain reaction (PCR), followed by the analysis of the amplifiedmolecules using techniques known to those skilled in the art. Suitableprimers can be routinely designed by one of skill in the art.

Hybridization and amplification techniques described herein may be usedto assay qualitative and quantitative aspects of expression of genesencoding endoglycan and/or podocalyxin. For example, RNA may be isolatedfrom a cell type or tissue known to express a gene encoding endoglycanand/or podocalyxin, and tested utilizing the hybridization (e.g.standard Northern analyses) or PCR techniques referred to herein. Thetechniques may be used to detect differences in transcript size whichmay be due to normal or abnormal alternative splicing. The techniquesmay be used to detect quantitative differences between levels of fulllength and/or alternatively splice transcripts detected in normalindividuals relative to those individuals exhibiting symptoms of acancer involving an endoglycan and/or podocalyxin protein or gene.

The primers and probes may be used in the above described methods insitu i.e. directly on tissue sections (fixed and/or frozen) of patienttissue obtained from biopsies or resections.

Accordingly, the present invention provides a method of detecting cancerin a patient comprising:

(a) extracting nucleic acid molecules comprising the podocalyxin gene orportion thereof from a sample from the patient;

(b) amplifying the extracted nucleic acid molecules using the polymerasechain reaction;

(c) determining the presence of nucleic acid molecules encodingpodocalyxin; and

(d) comparing the level of podocalyxin in the sample to a controlsample, wherein increased levels of podocalyxin as compared to thecontrol indicates that the patient has cancer.

In another embodiment, the present invention provides a method ofdetecting cancer in a patient comprising:

(a) extracting nucleic acid molecules comprising the endoglycan gene orportion thereof from a sample from the patient;

(b) amplifying the extracted nucleic acid molecules using the polymerasechain reaction;

(c) determining the presence of nucleic acid molecules encodingendoglycan; and

(d) comparing the level of endoglycan in the sample to a control sample,wherein decreased levels of endoglycan as compared to the controlindicates that the patient has cancer.

In a further embodiment, the present invention provides a method ofdetecting cancer in a patient comprising:

(a) extracting nucleic acid molecules comprising the endoglycan gene orportion thereof from the sample and the podocalyxin gene or portionthereof from a sample from the patient;

(b) amplifying the extracted nucleic acid molecules using the polymerasechain reaction;

(c) determining the presence of nucleic acid molecules encodingendoglycan and podocalyxin; and

(d) comparing the ratio of endoglycan to podocalyxin in the sample to acontrol sample, wherein a decreased ratio of endoglycan to podocalyxinas compared to the control indicates that the patient has cancer.

(b) Methods for Detecting Proteins

In another embodiment, the methods of the invention involve thedetection of the endoglycan and/or podocalyxin protein. In oneembodiment, the endoglycan protein is detected using antibodies thatspecifically bind to endoglycan and/or the podocalyxin protein isdetected using antibodies that specifically bind to podocalyxin.

Antibodies to the endoglycan and/or podocalyxin may also be preparedusing techniques known in the art. For example, by using a peptide of anendoglycan or podocalyxin, polyclonal antisera or monoclonal antibodiescan be made using standard methods. A mammal, (e.g., a mouse, hamster,or rabbit) can be immunized with an immunogenic form of the peptidewhich elicits an antibody response in the mammal. Techniques forconferring immunogenicity on a peptide include conjugation to carriersor other techniques well known in the art. For example, the protein orpeptide can be administered in the presence of adjuvant. The progress ofimmunization can be monitored by detection of antibody titers in plasmaor serum. Standard ELISA or other immunoassay procedures can be usedwith the immunogen as antigen to assess the levels of antibodies.Following immunization, antisera can be obtained and, if desired,polyclonal antibodies isolated from the sera.

To produce monoclonal antibodies, antibody producing cells (lymphocytes)can be harvested from an immunized animal and fused with myeloma cellsby standard somatic cell fusion procedures thus immortalizing thesecells and yielding hybridoma cells. Such techniques are well known inthe art, (e.g., the hybridoma technique originally developed by Kohlerand Milstein (Nature 256, 495-497 (1975)) as well as other techniquessuch as the human B-cell hybridoma technique (Kozbor et al., Immunol.Today 4, 72 (1983)), the EBV-hybridoma technique to produce humanmonoclonal antibodies (Cole et al. Monoclonal Antibodies in CancerTherapy (1985) Allen R. Bliss, Inc., pages 77-96), and screening ofcombinatorial antibody libraries (Huse et al., Science 246, 1275(1989)). Hybridoma cells can be screened immunochemically for productionof antibodies specifically reactive with the peptide and the monoclonalantibodies can be isolated.

The inventors have created a monoclonal antibody to endoglycan (Example2). Accordingly, in another embodiment, the endoglycan protein isdetected using a monoclonal antibody raised against a peptide having thesequence V A S M E D P G Q A P D L P N L P S I L P K M D L A E P P W H MP L Q G C (SEQ ID NO:10) that specifically binds to endoglycan.

The term “specifically binds to endoglycan” means reactivity againstendoglycan is clearly distinguishable from any reactivity against CD34or podocalyxin.

The term “specifically binds to podocalyxin” means reactivity againstpodocalyxin is clearly distinguishable from any reactivity against CD34or endoglycan.

The term “antibody” as used herein is intended to include fragmentsthereof which also specifically react with an endoglycan or fragmentsthereof or a podocalyxin or fragments thereof. Antibodies can befragmented using conventional techniques and the fragments screened forutility in the same manner as described above. For example, F(ab′)2fragments can be generated by treating antibody with pepsin. Theresulting F(ab′)2 fragment can be treated to reduce disulfide bridges toproduce Fab′ fragments.

Chimeric antibody derivatives, i.e., antibody molecules that combine anon-human animal variable region and a human constant region are alsocontemplated within the scope of the invention. Chimeric antibodymolecules can include, for example, the antigen binding domain from anantibody of a mouse, rat, or other species, with human constant regions.Conventional methods may be used to make chimeric antibodies containingthe immunoglobulin variable region which recognizes the gene product ofendoglycan/podocalyxin antigens of the invention (See, for example,Morrison et al., Proc. Natl Acad. Sci. U.S.A. 81,6851 (1985); Takeda etal., Nature 314, 452 (1985), Cabilly et al., U.S. Pat. No. 4,816,567;Boss et al., U.S. Pat. No. 4,816,397; Tanaguchi et al., European PatentPublication EP171496; European Patent Publication 0173494, UnitedKingdom patent GB 2177096B). It is expected that chimeric antibodieswould be less immunogenic in a human subject than the correspondingnon-chimeric antibody.

Monoclonal or chimeric antibodies specifically reactive with a proteinof the invention as described herein can be further humanized byproducing human constant region chimeras, in which parts of the variableregions, particularly the conserved framework regions of theantigen-binding domain, are of human origin and only the hypervariableregions are of non-human origin. Such immunoglobulin molecules may bemade by techniques known in the art, (e.g., Teng et al., Proc. Natl.Acad. Sci. U.S.A., 80, 7308-7312 (1983); Kozbor et al., ImmunologyToday, 4, 7279 (1983); Olsson et al., Meth. Enzymol., 92, 3-16 (1982)),and PCT Publication WO92/06193 or EP 0239400). Humanized antibodies canalso be commercially produced (Scotgen Limited, 2 Holly Road,Twickenham, Middlesex, Great Britain.)

Specific antibodies, or antibody fragments, such as, but not limited to,single-chain Fv monoclonal antibodies reactive against endoglycan orpodocalyxin may also be generated by screening expression librariesencoding immunoglobulin genes, or portions thereof, expressed inbacteria with peptides produced from the nucleic acid molecules ofendoglycan. For example, complete Fab fragments, VH regions and FVregions can be expressed in bacteria using phage expression libraries(See for example Ward et al., Nature 341, 544-546: (1989); Huse et al.,Science 246, 1275-1281 (1989); and McCafferty et al. Nature 348, 552-554(1990)). Alternatively, a SCID-hu mouse, for example the model developedby Genpharm, can be used to produce antibodies or fragments thereof.

Antibodies specifically reactive with endoglycan and/or podocalyxin, orderivatives, such as enzyme conjugates or labeled derivatives, may beused to detect endoglycan and/or podocalyxin in various samples (e.g.biological materials). They may be used as diagnostic or prognosticreagents and they may be used to detect abnormalities in the level ofprotein expression, or abnormalities in the structure, and/or temporal,tissue, cellular, or subcellular location of an endoglycan and/orpodocalyxin. In vitro immunoassays may also be used to assess or monitorthe efficacy of particular therapies. The antibodies of the inventionmay also be used in vitro to determine the level of expression of a geneencoding endoglycan and/or podocalyxin in cells genetically engineeredto produce an endoglycan and/or podocalyxin protein.

The antibodies may be used in any known immunoassays which rely on thebinding interaction between an antigenic determinant of endoglycanand/or podocalyxin and the antibodies. Examples of such assays areradioimmunoassays, enzyme immunoassays (e.g. ELISA), immunofluorescence,immunoprecipitation, latex agglutination, hemagglutination, andhistochemical tests. The antibodies may be used to detect and quantifyendoglycan and/or podocalyxin in a sample in order to determine its rolein cancer and to diagnose the cancer.

In particular, the antibodies of the invention may be used inimmuno-histochemical analyses, for example, at the cellular andsub-subcellular level, to detect an endoglycan protein and/or apodocalyxin protein, to localize it to particular cells and tissues, andto specific subcellular locations, and to quantitate the level ofexpression.

Cytochemical techniques known in the art for localizing antigens usinglight and electron microscopy may be used to detect endoglycan and/orpodocalyxin. Generally, an antibody of the invention may be labeled witha detectable substance and an endoglycan and/or podocalyxin protein maybe localised in tissues and cells based upon the presence of thedetectable substance. Examples of detectable substances include, but arenot limited to, the following: radioisotopes (e.g., ³H, ¹⁴C, ³⁵S, ¹²⁵I,¹³¹I), fluorescent labels (e.g., FITC, rhodamine, lanthanide phosphors),luminescent labels such as luminol; enzymatic labels (e.g., horseradishperoxidase, beta-galactosidase, luciferase, alkaline phosphatase,acetylcholinesterase), biotinyl groups (which can be detected by markedavidin e.g., streptavidin containing a fluorescent marker or enzymaticactivity that can be detected by optical or calorimetric methods),predetermined polypeptide epitopes recognized by a secondary reporter(e.g., leucine zipper pair sequences, binding sites for secondaryantibodies, metal binding domains, epitope tags). In some embodiments,labels are attached via spacer arms of various lengths to reducepotential steric hindrance. Antibodies may also be coupled to electrondense substances, such as ferritin or colloidal gold, which are readilyvisualised by electron microscopy.

The antibody or sample may be immobilized on a carrier or solid supportwhich is capable of immobilizing cells, antibodies etc. For example, thecarrier or support may be nitrocellulose, or glass, polyacrylamides,gabbros, and magnetite. The support material may have any possibleconfiguration including spherical (e.g. bead), cylindrical (e.g. insidesurface of a test tube or well, or the external surface of a rod), orflat (e.g. sheet, test strip). Indirect methods may also be employed inwhich the primary antigen-antibody reaction is amplified by theintroduction of a second antibody, having specificity for the antibodyreactive against endoglycan and/or podocalyxin protein. By way ofexample, if the antibody having specificity against endoglycan proteinis a rabbit IgG antibody, the second antibody may be goat anti-rabbitgamma-globulin labeled with a detectable substance as described herein.

Where a radioactive label is used as a detectable substance, endoglycanand/or podocalyxin may be localized by radioautography. The results ofradioautography may be quantitated by determining the density ofparticles in the radioautographs by various optical methods, or bycounting the grains.

Labeled antibodies against endoglycan and/or podocalyxin protein may beused in locating tumor tissue in patients undergoing surgery i.e. inimaging. Typically for in vivo applications, antibodies are labeled withradioactive labels (e.g. iodine-123, iodine-125, iodine-131, gallium-67,technetium-99, and indium-111). Labeled antibody preparations may beadministered to a patient intravenously in an appropriate carrier at atime several hours to four days before the tissue is imaged. During thisperiod unbound fractions are cleared from the patient and the onlyremaining antibodies are those associated with tumor tissue. Thepresence of the isotope is detected using a suitable gamma camera. Thelabeled tissue can be correlated with known markers on the patient'sbody to pinpoint the location of the tumor for the surgeon.

Accordingly, in another embodiment the present invention provides amethod for detecting cancer in a patient comprising:

(a) contacting a sample from the patient with an antibody that binds topodocalyxin;

(b) detecting the level of podocalyxin in a sample from the patient; and

(c) comparing the level of podocalyxin in the sample to a controlsample, wherein increased levels of podocalyxin as compared to thecontrol indicates that the patient has cancer.

In another embodiment the present invention provides a method fordetecting cancer in a patient comprising:

(a) contacting a sample from the patient with an antibody that binds toendoglycan;

(b) detecting the level of endoglycan in a sample from the patient; and

(c) comparing the level of endoglycan in the sample to a control sample,wherein decreased levels of endoglycan as compared to the controlindicates that the patient has cancer.

In a further embodiment, the present invention provides a method fordetecting cancer in a patient comprising:

(a) contacting a sample from the patient with a first antibody thatbinds to endoglycan and a second antibody that binds to podocalyxin;

(b) detecting the level of endoglycan and podocalyxin in the sample; and

(d) comparing the ratio of endoglycan to podocalyxin in the sample to acontrol sample, wherein a decreased ratio of endoglycan to podocalyxinas compared to the control indicates that the patient has cancer.

In a specific embodiment of the invention, breast tissue samples can bescreened using an anti-endoglycan antibody, such as the monoclonalantibody of Example 2 and/or an anti-podocalyxin antibody. Antibodybinding is detected using an appropriate detection system, preferablythe Envision detection system, and staining is scored based on theintensity of cellular staining and the proportion of cells stained.Tissue samples are designated “0” (strong endoglycan staining in themajority of tumor cells, and/or no discernable podocalyxin staining),“1” (a mixture of weak and intense membrane staining for endoglycanand/or podocalyxin), “2” (weak endoglycan, and/or strong podocalyxin,staining in the majority of tumor cells) or “3” (no discernableendoglycan staining, and/or high podocalyxin staining). Tissue samplesexhibiting no discernable endoglycan staining in the majority of tumorcells and/or high podocalyxin staining (designated “3”) have asignificantly poorer outcome when compared with the other threedesignations.

II. Kits

The methods described herein may be performed by utilizing pre-packageddiagnostic kits comprising the necessary reagents to perform any of themethods of the invention. For example, the kits may include at least onespecific nucleic acid or antibody described herein, which may beconveniently used, e.g., in clinical settings, to screen and diagnosepatients and to screen and identify those individuals exhibiting apredisposition to developing cancer. The kits may also include nucleicacid primers for amplifying nucleic acids encoding endoglycan and/orpodocalyxin in the polymerase chain reaction. The kits can also includenucleotides, enzymes and buffers useful in the method of the inventionas well as electrophoretic markers such as a 200 bp ladder. The kit willalso include detailed instructions for carrying out the methods of theinvention.

III. Therapeutic Methods

The finding by the present inventors that endoglycan and podocalyxin areinvolved in tumor progression allows the development of therapies totreat cancer including the identification of compounds that modulateendoglycan and/or podocalyxin. The present invention includes methods oftreating cancer by modulating, preferably activating or stimulating, thelevels of endoglycan on the cancer and/or preferably suppressing orinhibiting the levels of podocalyxin. The application also includesmethods for the identification of compounds that modulate the biologicalactivity of endoglycan and/or podocalyxin that may be used for thetreatment of cancers with decreased expression of endoglycan and/orincreased expression of podocalyxin.

Accordingly, the present invention provides a method of modulatingcancer cell growth by administering an effective amount of an agent thatmodulates endoglycan and/or podocalyxin to a cell or animal in needthereof. The present invention also provides a use of an agent thatmodulates endoglycan and/or podocalyxin to modulate cancer cell growth.The present invention further provides a use of an agent that modulatesendoglycan and/or podocalyxin in the manufacture of a medicament tomodulate cancer cell growth.

The terms “endoglycan”, “podocalyxin” and “cancer” as used herein are asdefined above in Section I.

The phrase “agent that modulates podocalyxin” includes any agent thatcan stimulate or activate podocalyxin (i.e. podocalyxin agonists) aswell as any agent that can inhibit or suppress podocalyxin (i.e.podocalyxin antagonists). Specific examples of podocalyxin modulatorsare given below.

The phrase “agent that modulates endoglycan” includes any agent that canstimulate or activate endoglycan (i.e. endoglycan agonists) as well asany agent that can inhibit or suppress endoglycan (i.e. endoglycanantagonists). Specific examples of endoglycan modulators are givenbelow.

The phrase “modulate cancer cell growth” as used herein refers to theinhibition or suppression as well as the activation or stimulation ofthe formation, differentiation, growth or development of cancer cells.

The phrase “effective amount” as used herein means an amount effective,at dosages and for periods of time necessary to achieve the desiredresults (e.g. the modulation of cancer cell growth). Effective amountsof a molecule may vary according to factors such as the disease state,age, sex, weight of the animal. Dosage regima may be adjusted to providethe optimum therapeutic response. For example, several divided doses maybe administered daily or the dose may be proportionally reduced asindicated by the exigencies of the therapeutic situation.

The term “animal” as used herein includes all members of the animalkingdom which express endoglycan and/or podocalyxin, preferably humans.

The term “a cell” includes a single cell as well as a plurality orpopulation of cells. Administering an agent to a cell includes both invitro and in vivo administrations.

In one aspect, the present invention provides a method of inhibitingcancer cell growth or treating cancer comprising administering aneffective amount of podocalyxin antagonist to a cell or animal in needthereof. The invention also provides a use of an effective amount ofpodocalyxin antagonist to inhibit cancer cell growth or treat cancer.The invention further provides a use of an effective amount ofpodocalyxin antagonist in the manufacture of a medicament to inhibitcancer cell growth or treat cancer.

In another aspect, the present invention provides a method of inhibitingcancer cell growth or treating cancer comprising administering aneffective amount of endoglycan agonist to a cell or animal in needthereof. The invention also provides a use of an effective amount ofendoglycan agonist to inhibit cancer cell growth or treat cancer. Theinvention further provides a use of an effective amount of endoglycanagonist in the manufacture of a medicament to inhibit cancer cell growthor treat cancer.

The phrase “inhibiting cancer cell growth” means that the growth of thecancer cell is decreased or reduced as compared to the growth of thecancer cell in the absence of the endoglycan agonist and/or podocalyxinantagonist.

The term “treatment or treating” as used herein means an approach forobtaining beneficial or desired results, including clinical results.Beneficial or desired clinical results can include, but are not limitedto, alleviation or amelioration of one or more symptoms or conditions,diminishment of extent of disease, stabilized (i.e. not worsening) stateof disease, preventing spread of disease, delay or slowing of diseaseprogression, amelioration or palliation of the disease state, andremission (whether partial or total), whether detectable orundetectable. “Treating” can also mean prolonging survival as comparedto expected survival if not receiving treatment.

In a preferred embodiment, the therapeutic methods of the invention areused to treat breast cancer.

The phrase “podocalyxin antagonist” means any agent that can inhibit orreduce the activity, function or levels of expression of podocalyxin ona cancer cell. Examples of podocalyxin antagonists include, but are notlimited to, an antibody, small molcule, peptide mimetic, an antisenseoligonucleotide to podocalyxin or any molecule or protein that cancrosslink podocalyxin on the surface of the tumor cell.

In one embodiment, the podocalyxin antagonist is a small molecule thatbinds to podocalyxin. Accordingly, the present invention provides amethod of treating cancer comprising administering an effective amountof an antagonist that can bind podocalyxin to a cell or animal in needthereof.

In another embodiment, the podocalyxin antagonist is an antibody thatbinds podocalyxin. The preparation of antibodies to podocalyxin aredescribed above in Section I and the same procedures can be used toprepare antibodies with therapeutic efficacy. In a preferred embodiment,the antibody will selectively bind a tumor specific isoform ofpodocalyxin but the isoform found on normal cells. Accordingly, thepresent invention provides a method of treating cancer comprisingadministering an effective amount of an antibody that can bindpodocalyxin to a cell or animal in need thereof. The invention alsoprovides a use of an effective amount of podocalyxin antibody to inhibitcancer cell growth or treat cancer. The invention further provides a useof an effective amount of podocalyxin antibody in the manufacture of amedicament to inhibit cancer cell growth or treat cancer. Coating cancercells with anti-podocalyxin antibodies may inhibit cell growth or induceapoptosis. In specific embodiments, the antibody could be coupled to atoxin that can cause the death of the cancer cell.

In another embodiment, the podocalyxin antagonist is an antisenseoligonucleotide that can modulate the expression and/or activity ofpodocalyxin on cancer cells.

The term “antisense oligonucleotide” as used herein means a nucleotidesequence that is complimentary to its target.

The term “oligonucleotide” refers to an oligomer or polymer ofnucleotide or nucleoside monomers consisting of naturally occurringbases, sugars, and intersugar (backbone) linkages. The term alsoincludes modified or substituted oligomers comprising non-naturallyoccurring monomers or portions thereof, which function similarly. Suchmodified or substituted oligonucleotides may be preferred over naturallyoccurring forms because of properties such as enhanced cellular uptake,or increased stability in the presence of nucleases. The term alsoincludes chimeric oligonucleotides which contain two or more chemicallydistinct regions. For example, chimeric oligonucleotides may contain atleast one region of modified nucleotides that confer beneficialproperties (e.g. increased nuclease resistance, increased uptake intocells), or two or more oligonucleotides of the invention may be joinedto form a chimeric oligonucleotide.

The phrase “endoglycan agonist” means any agent that can activate orstimulate the activity, function or levels of expression of endoglycanon a cancer cell. Examples of endoglycan agonists include, but are notlimited to, an antibody, small molecule, peptide mimetic, a nucleic acidencoding endoglycan or fragment thereof, or any molecule or protein thatcan antagonize podocalyxin on the surface of the tumor cell.

In one embodiment, the endoglycan agonist is a small molecule that bindsto endoglycan. Accordingly, the present invention provides a method oftreating cancer comprising administering an effective amount of anagonist that can bind endoglycan to a cell or animal in need thereof.

The nucleic acids of the present invention (for example, podocalyxinantisense oligonucleotides and nucleic acids encoding endoglycan andfragments thereof) may be ribonucleic or deoxyribonucleic acids and maycontain naturally occurring bases including adenine, guanine, cytosine,thymidine and uracil. The oligonucleotides may also contain modifiedbases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl, 2-propyland other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil,6-aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-haloadenine, 8-aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines,8-hydroxyl adenine and other 8-substituted adenines, 8-halo guanines,8-amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxylguanine and other 8-substituted guanines, other aza and deaza uracils,thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uraciland 5-trifluoro cytosine.

Other nucleic acids of the invention may contain modified phosphorous,oxygen heteroatoms in the phosphate backbone, short chain alkyl orcycloalkyl intersugar linkages or short chain heteroatomic orheterocyclic intersugar linkages. For example, the nucleic acid maycontain phosphorothioates, phosphotriesters, methyl phosphonates, andphosphorodithioates. In an embodiment of the invention there arephosphorothioate bonds links between the four to six 3′-terminus bases.In another embodiment phosphorothioate bonds link all the nucleotides.

The nucleic acid of the invention may also comprise nucleotide analogsthat may be better suited as therapeutic or experimental reagents. Anexample of a nucleotide analogue is a peptide nucleic acid (PNA) whereinthe deoxyribose (or ribose) phosphate backbone in the DNA (or RNA), isreplaced with a polyamide backbone which is similar to that found inpeptides (P. E. Nielsen, et al Science 1991, 254, 1497). PNA analogueshave been shown to be resistant to degradation by enzymes and to haveextended lives in vivo and in vitro. PNAs also bind stronger to acomplimentary DNA sequence due to the lack of charge repulsion betweenthe PNA strand and the DNA strand. Other nucleic acids may containnucleotides containing polymer backbones, cyclic backbones, or acyclicbackbones. For example, the nucleotides may have morpholino backbonestructures (U.S. Pat. No. 5,034,506). Nucleic acids may also containgroups such as reporter groups, a group for improving thepharmacokinetic properties of a nucleic acid, or a group for improvingthe pharmacodynamic properties of a nucleic acid. Nucleic acids may alsohave sugar mimetics.

The nucleic acids may be constructed using chemical synthesis andenzymatic ligation reactions using procedures known in the art. Thenucleic acids of the invention or a fragment thereof, may be chemicallysynthesized using naturally occurring nucleotides or variously modifiednucleotides designed to increase the biological stability of themolecules or to increase the physical stability of the duplex formedwith mRNA or the native gene e.g. phosphorothioate derivatives andacridine substituted nucleotides. The sequences may be producedbiologically using an expression vector introduced into cells in theform of a recombinant plasmid, phagemid or attenuated virus in whichsequences are produced under the control of a high efficiency regulatoryregion, the activity of which may be determined by the cell type intowhich the vector is introduced.

The nucleic acids may be introduced into tissues or cells usingtechniques in the art including vectors (retroviral vectors, adenoviralvectors and DNA virus vectors) or physical techniques such asmicroinjection. The nucleic acids may be directly administered in vivoor may be used to transfect cells in vitro which are then administeredin vivo. In one embodiment, the nucleic acids may be delivered tomacrophages and/or endothelial cells in a liposome formulation.

Peptide mimetics of endoglycan and/or podocalyxin may also be preparedas endoglycan modulators or agonists and/or podocalyxin modulators orantagonists. Such peptides may include competitive inhibitors,enhancers, peptide mimetics, and the like. All of these peptides as wellas molecules substantially homologous, complementary or otherwisefunctionally or structurally equivalent to these peptides may be usedfor purposes of the present invention.

“Peptide mimetics” are structures which serve as substitutes forpeptides in interactions between molecules (See Morgan et al (1989),Ann. Reports Med. Chem. 24:243-252 for a review). Peptide mimeticsinclude synthetic structures which may or may not contain amino acidsand/or peptide bonds but retain the structural and functional featuresof a peptide, or enhancer or inhibitor of the invention. Peptidemimetics also include peptoids, oligopeptoids (Simon et al (1972) Proc.Natl. Acad, Sci USA 89:9367); and peptide libraries containing peptidesof a designed length representing all possible sequences of amino acidscorresponding to an endoglycan peptide of the invention.

Peptide mimetics may be designed based on information obtained bysystematic replacement of L-amino acids by D-amino acids, replacement ofside chains with groups having different electronic properties, and bysystematic replacement of peptide bonds with amide bond replacements.Local conformational constraints can also be introduced to determineconformational requirements for activity of a candidate peptide mimetic.The mimetics may include isosteric amide bonds, or D-amino acids tostabilize or promote reverse turn conformations and to help stabilizethe molecule. Cyclic amino acid analogues may be used to constrain aminoacid residues to particular conformational states. The mimetics can alsoinclude mimics of inhibitor peptide secondary structures. Thesestructures can model the 3-dimensional orientation of amino acidresidues into the known secondary conformations of proteins. Peptoidsmay also be used which are oligomers of N-substituted amino acids andcan be used as motifs for the generation of chemically diverse librariesof novel molecules.

Peptides derived from endoglycan isoforms and/or podocalyxin isoformsmay also be used to identify lead compounds for drug development. Thestructure of the peptides described herein can be readily determined bya number of methods such as NMR and X-ray crystallography. A comparisonof the structures of peptides similar in sequence, but differing in thebiological activities they elicit in target molecules can provideinformation about the structure-activity relationship of the target.Information obtained from the examination of structure-activityrelationships can be used to design either modified peptides, or othersmall molecules or lead compounds that can be tested for predictedproperties as related to the target molecule. The activity of the leadcompounds can be evaluated using assays similar to those describedherein.

Information about structure-activity relationships may also be obtainedfrom co-crystallization studies. In these studies, a peptide with adesired activity is crystallized in association with a target molecule,and the X-ray structure of the complex is determined. The structure canthen be compared to the structure of the target molecule in its nativestate, and information from such a comparison may be used to designcompounds expected to possess the desired activity. Accordingly, in oneembodiment, endoglycan may be cocrystallized with podocalyxin and thestructure can then be compared to the structure of podocalyxin in itsnative state, to obtain information that may be used to design compoundsthat mimic endoglycan antagonism of podocalyxin.

IV. Screening Assays

The present invention also includes screening assays for identifyingagents that modulate endoglycan and/or podocalyxin and that are usefulin modulating cancer cell growth. Agents that modulate include agentsthat stimulate endoglycan and/or podocalyxin (endoglycan and/orpodocalyxin agonists) and agents that inhibit endoglycan and/orpodocalyxin (endoglycan and/or podocalyxin antagonists).

In accordance with one embodiment, the invention provides a method forscreening candidate compounds for their ability to modulate the activityof endoglycan and/or podocalyxin. The method comprises providing anassay system for assaying endoglycan and/or podocalyxin levels, assayingthe levels in the presence or absence of the candidate or test compoundand determining whether the compound has increased or decreasedendoglycan and/or podocalyxin levels.

Accordingly, the present invention provides a method for identifying acompound that modulates podocalyxin comprising:

(a) incubating a test compound with podocalyxin or a nucleic acidencoding podocalyxin; and

(b) determining the effect of the compound on podocalyxin activity orexpression and comparing with a control (i.e. in the absence of the testsubstance), wherein a change in the podocalyxin activity or expressionas compared to the control indicates that the test compound modulatespodocalyxin.

In another embodiment, the present invention provides a method foridentifying a compound that modulates endoglycan comprising:

(a) incubating a test compound with endoglycan or a nucleic acidencoding endoglycan; and

(b) determining the effect of the compound on endoglycan activity orexpression and comparing with a control (i.e. in the absence of the testsubstance), wherein a change in the endoglycan activity or expression ascompared to the control indicates that the test compound modulatesendoglycan.

The present invention also provides a screening assay that can be usedto identify endoglycan agonists and/or podocalyxin antagonists.

Accordingly, the present invention provides a screening assay foridentifying an antagonist of podocalyxin comprising the steps of:

(a) incubating a test substance with podocalyxin; and

(b) determining whether or not the test substance inhibits podocalyxinactivity, function or expression levels.

In another embodiment, the present invention provides a screening assayfor identifying an agonist of endoglycan comprising the steps of:

(a) incubating a test substance with endoglycan; and

(b) determining whether or not the test substance activates endoglycanactivity, function or expression levels.

The endoglycan and/or podocalyxin is generally immobilized in the aboveassays. Preferably, the endoglycan and/or podocalyxin is expressed onthe surface of a cell, more preferably a cancer cell.

Since endoglycan and podocalyxin both bind to NHERF, the invention alsoprovides a method for identifying a compound that modulates NHERFcomprising:

-   -   (a) incubating a test compound with NHERF or with cells        expressing NHERF on its surface; and    -   (b) determining the effect of the compound on NHERF activity or        expression and comparing with a control (i.e. in the absence of        the test substance), wherein a change in the NHERF activity or        expression as compared to the control indicates that the test        compound modulates NHERF. A change in NHERF activity may include        a change in response to endoglycan and/or podocalyxin.

Agents that modulate include agents that stimulate NHERF (NHERFagonists) and agents that inhibit NHERF (NHERF antagonists). In oneembodiment, the screening assay can be used to identify NHERFantagonists.

In all of the above screening assays, the test compound can be anycompound which one wishes to test including, but not limited to,proteins, peptides, nucleic acids (including RNA, DNA, antisenseoligonucleotides, peptide nucleic acids), carbohydrates, organiccompounds, small molecules, natural products, library extracts, bodilyfluids and other samples that one wishes to test for modulators ofendoglycan or NHERF.

One skilled in the art will appreciate that many methods can be used inorder to determine whether or not a test substance can activateendoglycan, inhibit podocalyxin or modulate NHERF and therefore inhibitcancer cell growth. Once a compound is identified in a screening assay(Endoglycan agonist, podocalyxin antagonist or NHERF modulator), it maybe tested in in vitro or in vivo assays to determine its effect oncancer cell growth.

The screening methods of the invention include high-throughput screeningapplications. For example, a high-throughput screening assay may be usedwhich comprises any of the methods according to the invention whereinaliquots of cells transfected with endoglycan and/or podocalyxin areexposed to a plurality of test compounds within different wells of amulti-well plate. Further, a high-throughput screening assay accordingto the invention involves aliquots of transfected cells which areexposed to a plurality of candidate factors in a miniaturized assaysystem of any kind. Another embodiment of a high-throughput screeningassay could involve exposing a transfected cell populationsimultaneously to a plurality of test compounds.

The method of the invention may be “miniaturized” in an assay systemthrough any acceptable method of miniaturization, including but notlimited to multi-well plates, such as 24, 48, 96 or 384-wells per plate,micro-chips or slides. The assay may be reduced in size to be conductedon a micro-chip support, advantageously involving smaller amounts ofreagent and other materials. Any miniaturization of the process which isconducive to high-throughput screening is within the scope of theinvention.

The invention extends to any compounds or modulators of endoglycanand/or podocalyxin identified using the screening method of theinvention that are useful in treating cancer.

The invention also includes a pharmaceutical composition comprising amodulator of endoglycan and/or podocalyxin identified using thescreening method of the invention in admixture with a suitable diluentor carrier. The invention further includes a method of preparing apharmaceutical composition for use in modulating cancer cell growthcomprising mixing a modulator of endoglycan and/or podocalyxinidentified according to the screening assay of the invention with asuitable diluent or carrier.

The present invention also includes all business applications of thescreening assay of the invention including conducting a drug discoverybusiness. Accordingly, the present invention also provides a method ofconducting a drug discovery business comprising:

(a) providing one or more assay systems for identifying a modulator ofpodocalyxin;

(b) conducting therapeutic profiling of modulators identified in step(a), or further analogs thereof, for efficacy and toxicity in animals;and

(c) formulating a pharmaceutical preparation including one or moremodulators identified in step (b) as having an acceptable therapeuticprofile.

In another embodiment, the present invention also provides a method ofconducting a drug discovery business comprising:

(a) providing one or more assay systems for identifying a modulator ofendoglycan;

(b) conducting therapeutic profiling of modulators identified in step(a), or further analogs thereof, for efficacy and toxicity in animals;and

(c) formulating a pharmaceutical preparation including one or moremodulators identified in step (b) as having an acceptable therapeuticprofile.

In certain embodiments, the subject method can also include a step ofestablishing a distribution system for distributing the pharmaceuticalpreparation for sale, and may optionally include establishing a salesgroup for marketing the pharmaceutical preparation.

The present invention also provides a method of conducting a targetdiscovery business comprising:

(a) providing one or more assay systems for identifying modulators ofpodocalyxin;

(b) (optionally) conducting therapeutic profiling of modulatorsidentified in step (a) for efficacy and toxicity in animals; and

(c) licensing, to a third party, the rights for further drug developmentand/or sales for modulators identified in step (a), or analogs thereof.

In another embodiment, the present invention provides a method ofconducting a target discovery business comprising:

(a) providing one or more assay systems for identifying modulators ofendoglycan;

(b) (optionally) conducting therapeutic profiling of modulatorsidentified in step (a) for efficacy and toxicity in animals; and

(c) licensing, to a third party, the rights for further drug developmentand/or sales for modulators identified in step (a), or analogs thereof.

V. Pharmaceutical Compositions

The present invention includes pharmaceutical compositions containingone or more modulators of endoglycan and/or podocalyxin. Accordingly,the present invention provides a pharmaceutical composition for use inmodulating cancer cell growth comprising an effective amount ofpodocalyxin modulator in admixture with a suitable diluent or carrier.In another embodiment, the present invention provides a pharmaceuticalcomposition for use in modulating cancer cell growth comprising aneffective amount of endoglycan modulator in admixture with a suitablediluent or carrier. In a further embodiment, the present inventionprovides a pharmaceutical composition for use in modulating cancer cellgrowth comprising an effective amount of endoglycan modulator andpodocalyxin modulator in admixture with a suitable diluent or carrier

In one embodiment, the present invention provides a pharmaceuticalcomposition for use in treating cancer comprising an effective amount ofa podocalyxin antagonist in admixture with a suitable diluent orcarrier. In another embodiment, the present invention provides apharmaceutical composition for use in treating cancer comprising aneffective amount of an endoglycan agonist in admixture with a suitablediluent or carrier. In a further embodiment, the present inventionprovides a pharmaceutical composition for use in treating cancercomprising an effective amount of an endoglycan agonist and apodocalyxin antagonist in admixture with a suitable diluent or carrier.

Such pharmaceutical compositions can be for intralesional, intravenous,topical, rectal, parenteral, local, inhalant or subcutaneous,intradermal, intramuscular, intrathecal, transperitoneal, oral, andintracerebral use. The composition can be in liquid, solid or semisolidform, for example pills, tablets, creams, gelatin capsules, capsules,suppositories, soft gelatin capsules, gels, membranes, tubelets,solutions or suspensions. The endoglycan and/or podocalyxin or ligand ispreferably injected in a saline solution either intravenously,intraperitoneally or subcutaneously.

The pharmaceutical compositions of the invention can be intended foradministration to humans or animals. Dosages to be administered dependon individual needs, on the desired effect and on the chosen route ofadministration.

The pharmaceutical compositions can be prepared by per se known methodsfor the preparation of pharmaceutically acceptable compositions whichcan be administered to patients, and such that an effective quantity ofthe active substance is combined in a mixture with a pharmaceuticallyacceptable vehicle. Suitable vehicles are described, for example, inRemington's Pharmaceutical Sciences (Remington's PharmaceuticalSciences, Mack Publishing Company, Easton, Pa., USA 1985).

On this basis, the pharmaceutical compositions include, albeit notexclusively, the active compound or substance in association with one ormore pharmaceutically acceptable vehicles or diluents, and contained inbuffered solutions with a suitable pH and iso-osmotic with thephysiological fluids. The pharmaceutical compositions may additionallycontain other anti-cancer agents.

A pharmaceutical composition comprising the nucleic acid molecules ofthe invention may be used in gene therapy to treat cancer. Recombinantmolecules comprising a nucleic acid sequence encoding endoglycanmolecule of the invention, or fragment thereof or an antisensepodocalyxin molecule or fragment thereof, may be directly introducedinto cells or tissues in vivo using delivery vehicles such as retroviralvectors, adenoviral vectors and DNA virus vectors. They may also beintroduced into cells in vivo using physical techniques such asmicroinjection and electroporation or chemical methods such ascoprecipitation and incorporation of DNA into liposomes. Recombinantmolecules may also be delivered in the form of an aerosol or by lavage.The nucleic acid molecules of the invention may also be appliedextracellularly such as by direct injection into cells.

The following non-limiting examples are illustrative of the presentinvention:

EXAMPLES Example I Podocalyxin

Materials and Methods

Tissue Microarray Construction

A total of 270 formalin-fixed, paraffin-embedded primary invasive breastcancer tissue blocks (archival cases from Vancouver General Hospitalfrom the period 1974-1995) that had been graded according to theNottingham modification of the Scarth, Bloom, Richardson method (Elstonand Ellis, 1991) were used to construct a tissue microarray (TMA) asdescribed previously (Parker et al., 2002). Briefly, a tissue-arrayinginstrument (Beecher Instruments, Silver Springs Md.) was used to createholes in a recipient block with defined array coordinates. Two 0.6 mmdiameter tissue cores were taken from each case and transferred to therecipient block using a solid stylet. Three composite high-densitytissue microarray blocks were designed and serial 4 μm sections werethen cut with a microtome and transferred to adhesive-coated slides.Normal breast and kidney tissues were used as controls.

TMA Immunohistochemistry, Scoring and Correlation Analysis

Array and control tissue sections were deparaffinized and treated for 30min at 90° C. with citrate buffer (pH 6.00) for antigen retrieval. Thesections were then treated with 3% hydrogen peroxide in PBS for 30 minfollowed by incubation with the mouse monoclonal anti-human podocalyxinantibody 3D3 (1:80 dilution in 1% BSA in PBS; Kershaw et al., 1997a)overnight. Antibody binding was detected using the Envision detectionsystem (Dako) and the sections were then counterstained withhematoxylin, dehydrated and mounted.

Staining of the TMA sections was scored semi-quantitatively based on theintensity of cytoplasmic staining and the proportion of cells stained:0—no specific staining in the tumor cells; 1—diffuse, weakimmunoreactivity or strong cytoplasmic staining reaction in <10% of thetumor cells; 2—diffuse intermediate immunoreactivity or strongcytoplasmic staining in 10-50% of cells; 3—strong cytoplasmic stainingin >50% of the tumor cells. In the case of discrepancy between two coresfrom the same tumor sample, the higher score was used. All samples wereevaluated and scored independently without knowledge of the patient'soutcome information.

All scores were entered into a standardized Excel spreadsheet andprocessed using the software TMA-deconvoluter 1.06, Cluster and TreeViewprograms as previously described (Liu et al., 2002). Survival analysiswas performed using the Kaplan-Meier method. Paired correlation analysisto nodal status, grade, size and p53, ER, PR, and HER2 status, all ofwhich were previously assessed on the TMA (Parker et al., 2002, Liu etal., 2002; Makretsov et al., 2003) was performed using the bivariatetwo-tailed Pearson test. Multivariate survival analysis was performedusing the Cox proportional hazard regression model. Differences wereconsidered significant at p<0.05.

Cell Culture, Transfection and Podocalyxin Localization

T47D, MCF-7 and MDA-231 human breast cancer cell lines were maintainedin DMEM/F12 medium supplemented with 5% FBS (Hyclone) and insulin (5mg/ml). Endogenous podocalyxin expression was determined by Westernblotting of whole cell lysates (20 μg total protein) using the antibodydescribed above for the tissue array analysis.

MCF7 cells, which expressed low levels of endogenous human podocalyxin(see FIG. 2A) were transfected with a control empty pIRES-EGFPexpression vector (BD biosciences) or with the same vector containing afull length mouse podocalyxin cDNA inserted into the multiple cloningsite (BD Biosciences) using DMRIE-C reagent (Life Technologies/BRL).Stable transfectants were generated by continuous selection under G418(500 μg/ml; Life Technologies/BRL). Successful transfection wasdetermined by EGFP expression which, as expected, was heterogenous giventhat the transfectants were uncloned pools. Podocalyxin transgeneexpression, (which was also heterogeneous) was determined byimmunofluorescence of confluent monolayers using an antibody specificfor mouse podocalyxin (Doyonnas et al., 2001). The precise subcellularlocalization of the mouse podocalyxin was determined by confocalmicroscopy after dual staining of either the adherens junction proteinE-cadherin (mouse monoclonal, Pharmingen, San Diego Calif.) or of thetight junction proteins occludin and ZO-1 (mouse and rat monoclonalsrespectively, Zymed, San Francisco Calif.). Here the heterogenous natureof the pooled populations was useful as it clearly demarcated consistentdifferences in the cell junctions of podocalyxin expressing cells.

Results

Podocalyxin Expression is Weak to Negative in Normal Breast Tissue

Normal kidney sections were immunostained with anti-human podocalyxin asa positive control for antibody specificity (Kershaw D B et. al.,1997a). As expected, podocalyxin was highly expressed on glomerularpodocytes cells while expression was low to negative on tubular cells(FIG. 1A). This confirmed the specificity of immunocytochemical stainingunder the conditions used. Podocalyxin was also present in normal breasttissue but its expression was limited and it was spatially restricted.Specifically, podocalyxin was localized to the apical-most border inluminal epithelial cells (FIG. 1B; arrows). In addition, podocalyxin waspresent on the apical face of vascular endothelial cells as has beendescribed previously (FIG. 1A, B; arrowheads, Kershaw et al.1995,McNagny et al. 1997).

Podocalyxin is Expressed by Invasive Breast Carcinoma

To determine whether podocalyxin is upregulated by neoplastic breasttissue, an array of breast tissue samples was screened using ananti-podocalyxin antibody as probe. The clinicopathologicalcharacteristics of the 270 cases that made up the tissue microarray(TMA) are shown in Table 1. Sixty-one percent (165/270) of the invasivebreast carcinoma cases on the TMA exhibited no discernable podocalyxinstaining and were given a designation of ‘0’ (FIG. 1C). Twenty-threepercent (61/270) of the cases on the TMA exhibited weak staining in themajority of the tumor cells and they were given a designation of ‘1’(FIG. 1D). Eleven percent (31/270) of the cases exhibited a mixture ofweak and intense -membrane staining (FIG. 1E).These three groups couldnot be distinguished from each other on the basis of clinical outcome.Specifically, Kaplan-Meier analysis of the overall survival (data notshown) and disease free survival (FIG. 2A) indicated that these threeclassifications were indistinguishable in terms of outcome.

Five percent (13/270) of the cases on the TMA exhibited a strongstaining in the majority of the tumor cells and were originally given adesignation of ‘3’ (FIG. 1F). This designation had a significantlypoorer outcome compared to the other three original designations asassessed by Kaplan Meier curve analysis (FIG. 2A; p<0.02). Therefore,this difference was statistically significant and readily observablewhen the 0, 1, and 2 designations were grouped and described as ‘low orno podocalyxin’ and compared to designation 3 described as ‘highpodocalyxin’ (FIG. 2B p<0.02). In addition, the high podocalyxin tumorshad a mean survival time of 9.5+/−1.9 years, which was significantlyshorter than the mean survival time of 15+/−0.5 years for the combinedlow or no podocalyxin tumors. It was concluded that high levelexpression of podocalyxin is selective to the most metastatic tumors.

High Podocalyxin Expression is an Independent Marker of Poor Outcome

The same TMA that was used for podocalyxin staining has been previouslystained for a number of markers that have prognostic significance forbreast cancer outcome (Makretsov et al., Submitted and seepathology.ubc.ca/immuno). Thus, the inventors were able to perform amulti-variant Cox regression analysis in which high podocalyxinexpression was compared with 6 other breast cancer-associated markers(Table 2). As expected, nodal status and HER2 overexpression wereindependent markers of poor outcome, which is an internal validation ofthe array analysis. Therefore, the fact that high podocalyxin expressionon its own was associated with increased relative risk (p<0.006)indicates that it is an independent progonostic indicator of pooroutcome. Interestingly, however, a Pearson correlation analysis of thesame data indicated that high podocalyxin expression positivelycorrelated with p53 mutations, Estrogen receptor loss, and increasedtumor grade (Table 3; all p values <0.01). Thus, the data suggest thatpodocalyxin is an independent marker of metastatic tumors.

Ectopic Podocalyxin Expression leads to Disruption of Tight Junctionsand delamination of MCF-7 breast tumor cells

Previously it has been shown that ectopic expression of podocalyxin inkidney epithelial cells (MDCK), leads to disruption of cell junctions(Takeda et al., 2000). To determine if the same is true of breastcarcinoma cells the inventors first examined endogenous levels ofpodocalyxin in human breast tumor lines. Specifically, MCF-7 and T-47Dcells, which both are capable of forming cell junctions and morphogenicstructures, expressed low levels of endogenous human podocalyxincompared to the high levels of expression in the highly invasive andmetastatic MDA231 cells which do not form cell junctions (FIG. 3A). Totest the functional significance of this expression, human MCF-7 cellswere transfected with a control EGFP-expressing vector or the samevectorencoding EGFP and a full-length mouse podocalyxin. After selectiondrug resistance, the morphology of pooled heterogeneous populations ofprimary transfectants was examined. Control monolayers formed flatconfluent monolayers that were undistinguishable from the parent line(data not shown). In contrast, pooled populations stably transfectedwith the EGFP/Podocalyxin vector contained areas where cells bulgedoutward from the monolayers (FIG. 3B). As these cultures reachedconfluence they often shed podocalyxin-expressing cells into the media.Coordinate, yet heterogeneous, expression of EGFP and mouse podocalyxinwas confirmed by dual green channel fluorescence and immunostaining(FIG. 3B). Note also that podocalyxin was appropriately targeted to theapical membrane domain in the transfected cells (FIG. 3B lower panel).

Attempts to subclone high podocalyxin expressing cells failed as thesecells were constantly shed from the substratum and were difficult tomaintain in suspension. The inventors therefore attempted to more fullyanalyze the heterogeneous pooled populations produced in the primarytransfections. This allowed the effects of heterogeneous podocalyxinoverexpression on cell junctions to be analyzed by dual immunostaining.Interestingly, cells expressing low to negligible levels of thepodocalyxin transgene formed normaladherens junctions with the expectedbasolateral expression of E-cadherin and apical expression of the tightjunction protein, occludin along the lateral membranes at sites ofcell-cell interaction (FIG. 3C). In contrast, E-cadherin and occludinboth became widely distributed on the entire surface of highlyoverexpressing podocalyxin expressing cells (FIG. 3C). The latter cellswere clearly being extruded from the monolayers as evidenced by theirmorphology and upward migration of their DAPI-stained nuclei. These datasuggested that high levels of Podocalyxin expression can disrupt tightjunction-dependent apical/basal polarity in mammary carcinoma cells.This conclusion was further supported by the finding thattransepithelial resistance, which is a functional measure of tightjunctions, was reduced from 497+/−37.2 ohms/cm2 in control-transfectedMCF-7 monolayers to 210+/−11.9 ohms/cm2 in EGFP/Podocalyxin-transfectedmonolayers. Upexpression of podocalyxin in breast carcinoma cell linesleads to the disruption of cell-cell junctional complexes,mislocalization of cadherins and occludins and delamination frombasement membranes, all features common to more aggressive forms ofmetastatic breast cancer.

Example 2 Endoglycan

Results

Tissue Distribution of CD34 Family Members

Data was compiled from published analyses on human and mouse CD34,Podocalyxin and Endoglycan (Krause 1996, McNagny 1997, Doyonnas 2001,Sassetti 2000) and from our unpublished observations on mouseEndoglycan. Endoglycan and Podocalyxin expression profiles weregenerated using unpublished data obtained from: 1) Northern blots ofhematopoietic lineage cell lines, 2) RT-PCR of sorted hematopoieticsubsets from bone marrow, 3) antibody stains and flow cytometry analysisusing existing antibodies to CD34 (RAM34) Podocalyxin (PCLP1) and 4)Immunohistochemistry using the same antibodies. Results are shown inTable 4.

Preparation of Monoclonal Antibody with specific binding againstEndoglycan

To make the rat monoclonal antibody, rats were immunized with a peptidecorresponding to sequence from the extracellular domain: V A S M E D P GQ A P D L P N L P S I L P K M D L A E P P W H M P L Q G G C (SEQ ID NO:10) linked to KLH and boosted with the entire extracellular domain fusedto the Fc portion of Rabbit IgG1. Hybridomas were made using standardprotocols and antibodies from these hybridomas were screened forreactivity with the peptide and Fc-fusion protein by ELISA. They werealso screened for the ability to stain a rat myeloma cell line, Y3,which had been transfected to express full length Endoglycan. Oneantibody passed all criteria (F4B10). This antibody did not react withY3 cells expressing CD34 or Podocalyxin so the antibody is specific forEndoglycan and not related family members (FIG. 6). In addition, thisantibody reacts with mouse and human Endoglycan and so it may be auseful reagent for both species.

Expression of Endoglycan in Relation to Podocalyxin

Endoglycan and Podocalyxin have a mirror image pattern of expression inbreast cancer cell lines (FIG. 7). In MDA-231: metastatic tumor linewhere cells are non-polarized, Podocalyxin expression is high, whereasEndoglycan expression is negative. In MCF-7, a relatively non-metastaticline, cells maintain normal polarity, Podocalyxin expression is low,whereas Endoglycan is highly expressed. In T47D: a relativelynon-metastatic line, cells maintain normal polarity, Podocalyxinexpression is low, whereas Endoglycan expression is high. This wasdetermined by indirect immunofluorescence using our new antibody andflow cytometry (FACS).

Function of Endoglycan:

Despite Endoglycan's similarity to CD34 and Podocalyxin, it may have adifferent function. Endoglycan was expressed in CD34/CD43 deficient mastcells. Pure mast cell cultures can be obtained by culturing mouse bonemarrow in IL-3 for >4 weeks. Although normal mast cells grow in singlecell suspensions, mast cells grown from CD34/CD43 KO mice tend to formlarge aggregates. Infection of mast cells with a retrovirus expressingectopic CD34 reverses this aggregation and suggests that the normalfunction of CD34 is to block adhesion. In side by side experiments,ectopic expression of Endoglyean had no effect suggesting that it doesnot block adhesion and may instead have a pro-adhesive function. (FIG.8).

Discussion

The present inventors have demonstrated that abnormally high podocalyxinexpression and low endoglycan expression is a novel prognostic indicatorof poor outcome in invasive breast carcinoma.

Tissue microarrays afford investigators the opportunity to carry out arapid and relatively thorough screening of molecules that are believedto be important in specific tissues or pathologies (Kononen et al.,1998). The power of this technology is exemplified here where only 13 ofthe 270 cases on our TMA had uniformally high podocalyxin expression andyet this is clearly informative with respect to prognostic outcome. Theinventors are currently assembling a 3000 case invasive breast cancerTMA linked to treatment and outcome that should allow this resolvingpower to be increased significantly and evaluate the role of differenttherapies on podocalyxin status of tumors.

Locally invasive breast cancers can have markedly different treatmentresponses and outcomes. Thus, it is, extremely difficult to predictwhich patients will most benefit, or not benefit, from adjuvant therapy(Eifel et al., 2001). Genome-wide searches and large-scale expressionprofiling followed by cluster analysis have had some impact on thisproblem (Polyak et al., 2002), particularly with respect to identifyingthose tumors that do not progress (van't Veer et al., 2002). Despitethese advances, the identification of novel independent indicators ofpoor outcome continues to be useful, even if they are only important ina small proportion of tumors, because they facilitate the development ofnew classification parameters that increase the resolving power of highthroughput genomic and expression approaches. In addition, if thesemarkers play a functional role in the biology of metastatic progressionthey may be rational therapeutic targets and further experimentalinvestigations may lead to the discovery of other functionally relevantmolecules in progression. This has clearly been proven to be the casewith erbB2 (Nabholtz and Slamon, 2001).

CD34 and podocalyxin, expressed by high endothelial venules (HEV) aredecorated with the appropriate glycosylations to make them adhesiveligands for L-selectin expressed by circulating lymphocytes. This typeof posttranslational modification is exquisitely tissue-specific and thevast majority of endothelial cells and hematopoietic cells expressingCD34 type proteins lack this modification. On all other cell types, thedata suggest that these molecules serve as blockers of adhesion viatheir bulky, negatively-charged mucin domains, as has been demonstratedby both loss- and gain-of-function experiments (Doyonnas et al. 2001 andTakeda et al. 2000). The experiments described here clearly delineate ananti-adhesive role for podocalyxin.

Initial functional experiments suggest that forced podocalyxinover-expression disrupts tight junctions in well-behaved MCF-7 breastcarcinoma cells. Specifically, transepithelial resistance, a functionalindicator of tight junction patency was significantly reduced and thespatially-restricted tight junction-associated protein occludin becamevery diffusely localized. Moreover, it was found that the tightjunction-associated, PDZ domain-containing protein ZO-1 was mislocalizedand relocalized basally in podocalyxin expressing cells (data notshown). These observations indicate that podocalyxin can function as ananti-adhesive molecule in breast cancer cells and they agree withprevious findings in kidney epithelial cells where podocalyxinoverexpression was shown to disrupt tight junction function and proteinlocalization (Takeda et al., 2000) in vitro and podocalyxin loss wasshown to lead to inappropriate tight-junction maintenance in vivo. Infuture experiments it will be interesting to determine if the potentialPDZ-binding site at the extreme C-terminus of the podocalyxincytoplasmic domain (Doyonnas et al., 2001; Takeda et al., 2001)contributes to this disruption of the tight junction. As this site alsocontributes to the association of podocalyxin with the actincytoskeleton it may be involved in the cytoplasmic mislocalization ofthe protein itself that we observed in high expressing breast tumors(see FIG. 1F).

The adherens junction protein E-cadherin is often downregulated inlobular breast carcinomas but not in the much more prevalent ductalforms of the disease. Forced expression of podocalyxin did not cause aloss of E-cadherin expression in MCF-7 cells. Instead, it altered itslocalization. Specifically, E-cadherin remained at the membrane butrather than being restricted to the basolateral domain the adherensjunction protein was found along the entire circumference of highpodocalyxin expressing cells that were being extruded from the MCF-7monolayers. This could explain the somewhat paradoxical observation thatcircumferential E-cadherin localization is associated with poor outcomein grade III ductal breast carcinomas (Gillet et al., 2001). It alsosuggests that high podocalyxin expression may be disrupting apical-basalpolarity in breast epithelial cells, which is also one function ofabnormal erbB2 signaling (Brugge). A loss of polarity has been assumedto be functionally important in breast carcinoma progression, but thispossibility has not yet been formally tested (Roskelley and Bissell,2002). The inventors are currently carrying out such experiments using a3-dimensional model of normal, polarized mammary epithelial cellmorphogenesis (Roskelley et al., 2000).

Although a detailed dissection of the podocalyxin promoter regulatoryelements has not yet been performed, it has recently been shown to be adirect transcriptional target of the Wilm's Tumor suppressor protein,WT1 (Palmer R E et al. Current Biology 2001). The role of WT1 in tumorprogression is, at present, contentious. A tumor suppressive effect ofthis protein is supported by its loss in renal tumors and its ability toinduce differentiation and cell cycle arrest of kidney and hematopoieticlineage cells. On the other hand, upregulation of WT1 expression isfrequently observed in acute myeloid and lymphoid leukemias. Anexplanation for this apparent paradox could be the disrupted circuitryin tumor cells. For example WT1 may induce both a differentiation andcell cycle arrest program in normal cells, whereas tumor cells may havebecome refractory to the cell cycle arrest and only expressdifferentiation antigens like podocalyxin.

Since Endoglycan and Podocalyxin have very similar sequences in thecytoplasmic domain, they may be natural antagonists of each other:Endoglycan may promote adhesion, maintain cell polarity, and blockmetastasis, and Podocalyxin may block adhesion and decrease cellpolarity and increase metastasis. One theory is that endoglycan andpodocalyxin compete for binding to NHERF1; a molecule that haspreviously been shown to link Podocalyxin to the the actin cytoskeleton(Takeda et al., 2001). This then would allow these molecules (withopposing functions) to compete for localization in adhesion structures.

While the present invention has been described with reference to whatare presently considered to be the preferred examples, it is to beunderstood that the invention is not limited to the disclosed examples.To the contrary, the invention is intended to cover variousmodifications and equivalent arrangements included within the spirit andscope of the appended claims.

All publications, patents and patent applications are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety.

TABLE 1 Tissue Microarray Population Characteristics. Lymph node status:Negative  160 (66.9%) Positive   79 (29.3%) Unknown   31 (11.5%) Tumorgrade: 1   55 (20.4%) 2  148 (54.8%) 3   67 (24.8%) Tumor Size <10 mm  20 (7.4%)   10 mm-20 mm   43 (15.9%) >20 mm   72 (26.7%) Unknown  135(50%) Overall Survival Mean 14.9 years Median 15.0 years

TABLE 2 Cox Regression Multi-Variant Analysis 95% Confidence Degree ofSignificance Relative Interval for RR Marker Freedom (p)* Risk (RR)Lower Upper Podocalyxin 1 0.006 7.271 1.747 30.255 P53 1 0.121 2.7940.764 10.222 ER** 1 0.541 0.866 0.547 01.372 HER2 1 0.008 4.661 1.48514.624 Nodes 1 0.003 3.688 1.581 08.601 Grade 2 0.257 3.088 0.798 11.946Tumor Size 2 0.482 1.115 0.475 02.620 *Correlation is significant at the0.05 level. **PR gives the same result.

TABLE 3 Pearson Correlation Analysis Between Podocalyxin and Other KnownClinicohistopathological Markers. Pearson Number Marker CorrelationSignificance of Cases Podocalyxin   1.0 — 270 p53   0.180 0.006 236 ER−0.214 0.001 240 HER2 −0.032 0.613 258 Nodes −0.069 0.285 239 Grade  0.191 0.002 270

TABLE 4 Tissue Distribution of CD34 Family Members Tissue/CellsEndoglycan Podocalyxin CD34 Multipotent hematopoetic precursorsAdult + + + Embryo + + + Monopotent precursors Erythroid + + −Thrombocytic ? + + Myeloid +/− − + Lymphoid (subset of thymocytes)+? + + Mature hematopoetic cells B Cells (LPS activated) + − − T Cells −− − Macrophages − − − Granulocytes − − − Eosinophils − − − Mast Cells −− + Erythrocytes +* +* − Platelets ? + − Vessels Vascular endothelial− + + Vascular smooth muscle + − − Intestinal Epithellal + − − Podocytes+/− + − Brain (Neurons) + +** ? Boundary Elements (mesothellal) − + −*embryonic erythrocytes only **eppendymal layer only

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1. A method of determining whether or not a patient having breast orovarian cancer has a poor outcome comprising: (a) detecting the level ofpodocalyxin in a sample from the patient; and (b) comparing the level ofpodocalyxin in the sample to a control sample from non-metastaticcancer, wherein an increased level of podocalyxin as compared to thecontrol indicates that the patient having said breast or ovarian cancerhas a poor outcome.
 2. A method according to claim 1 wherein the canceris breast cancer.
 3. A method according to claim 1 or 2 whereindetermining the level in step (a) comprises determining the amount ofnucleic acid molecules.
 4. A method according to claim 3 wherein thenucleic acid molecules are mRNA.
 5. A method according to claim 1 or 2wherein determining the level in step (a) comprises determining theamount of protein.
 6. A method according to claim 5 wherein an antibodyis used to determine the amount of the protein.